13C-Labeled-Starch Breath Test in Congenital Sucrase-isomaltase Deficiency.

BACKGROUND AND HYPOTHESES: Human starch digestion is a multienzyme process involving 6 different enzymes: salivary and pancreatic α-amylase; sucrase and isomaltase (from sucrose-isomaltase [SI]), and maltase and glucoamylase (from maltase-glucoamylase [MGAM]). Together these enzymes cleave starch to smaller molecules ultimately resulting in the absorbable monosaccharide glucose. Approximately 80% of all mucosal maltase activity is accounted for by SI and the reminder by MGAM. Clinical studies suggest that starch may be poorly digested in those with congenital sucrase-isomaltase deficiency (CSID). Poor starch digestion occurs in individuals with CSID and can be documented using a noninvasive C-breath test (BT). METHODS: C-Labled starch was used as a test BT substrate in children with CSID. Sucrase deficiency was previously documented in study subjects by both duodenal biopsy enzyme assays and C-sucrose BT. Breath CO2 was quantitated at intervals before and after serial C-substrate loads (glucose followed 75 minutes later by starch). Variations in metabolism were normalized against C-glucose BT (coefficient of glucose absorption). Control subjects consisted of healthy family members and a group of children with functional abdominal pain with biopsy-proven sucrase sufficiency. RESULTS: Children with CSID had a significant reduction of C-starch digestion mirroring that of their duodenal sucrase and maltase activity and C-sucrase BT. CONCLUSIONS: In children with CSID, starch digestion may be impaired. In children with CSID, starch digestion correlates well with measures of sucrase activity.

Characteristics of cardiac epithelium at the esophagogastric junction of a pediatric population with gastroesophageal reflux.

Cardiac mucosa (CM) of the adult, regardless its location, shares phenotypic characteristics with Barrett's epithelium, namely villin expression and a Barrett's pattern of cytokeratins 7 and 20 expression. As far as we know, the phenotypic profile of CM in children has not been studied. The objective was to evaluate the phenotypic profile of cardiac mucosa from the esophagogastric junction of children with reflux symptoms. Biopsies routinely performed at the esophagogastric junction of children submitted to upper-gastrointestinal endoscopy for complaints suggestive of reflux were retrieved from the archive and used for the purposes of this study. Biopsies were assessed for the presence of squamous epithelium, cardiac and oxyntic mucosa and intestinal metaplasia. Samples displaying both squamous and columnar epithelia were immunohistochemically evaluated for the presence of villin and sucrase-isomaltae and for the expression of cytokeratins 7 and 20. From the 42 biopsies samples retrieved, 30 had simultaneously squamous and columnar epithelia. Cardiac mucosa was present in 86.7% of the cases, and intestinal metaplasia was observed only in one (3.3%). Villin expression in cardiac mucosa was observed in 96% of the cases and a cytokeratins 7 and 20 Barrett's pattern in 73%. Sucrase-isomaltase and MUC2 were only expressed in the case with intestinal metaplasia. Cardiac mucosa was high prevalent in biopsies from the esophagogastric junction of children with reflux. As in adults, cardiac mucosa in children has an immunoprofile similar to Barrett's esophagus. For the first time, it was shown that pediatric cardiac mucosa frequently expresses villin.

Mosaic pattern of sucrase isomaltase deficiency in two brothers.

The pathophysiology of mucosal changes observed in infants with chronic protracted diarrhea is poorly understood. We report on two brothers suffering from a special form of sucrase isomaltase (SI) deficiency. The children presented with weight loss and dyspepsia after sucrose exposition. We performed an H respiration test, which showed a pathologic result in the younger brother. Analysis of the brush border enzyme activities showed low expression of lactase and SI. Immunoelectron microscopy of duodenal biopsies showed an isolated SI deficiency in a mosaic pattern [e.g., 42% (14%) crypt enterocytes and 64% (59%) villus enterocytes with decreased amounts of SI on microvilli], whereas lactase and aminopeptidase n (ApN) were present at the apical membrane of all cells in a normal range. The SI mosaic pattern of these patients shows that the enterocytes contain low amounts of SI on the apical membrane but express normal quantities of other disaccharidases. These findings suggest the existence of different clonal expressions or specific (posttranslational) mechanisms of postGolgi transportation for individual brush border enzymes. It remains unresolved whether the mosaic distribution is part of a normal maturation process or caused by a lack of an overall control mechanism in the expression of brush border hydrolases.

Congenital sucrase-isomaltase deficiency because of an accumulation of the mutant enzyme in the endoplasmic reticulum.

BACKGROUND & AIMS: Congenital sucrase-isomaltase deficiency (CSID) is an autosomal recessive human disorder characterized by reduced activities of the brush border enzyme sucrase-isomaltase (SI). Here, we elucidate the pathogenesis of a new variant of CSID at the cellular and molecular level. METHODS: Assessment of the CSID phenotype was achieved by enzymatic activity measurements, biosynthetic labeling of intestinal biopsy specimens, immunoprecipitation of SI, and immunoelectronmicroscopy. The putative mutation was identified by sequencing of the SI cDNA isolated by RT-PCR from intestinal biopsy samples. The function of the mutation was verified by immunoprecipitation and confocal microscopy of transiently transfected cells. RESULTS: Biosynthetic labeling and immunoelectron microscopy reveal a predominant localization of SI in the endoplasmic reticulum (ER) similar to phenotype I of CSID. Unlike phenotype I, however, a partial conversion of SI to a complex glycosylated mature form takes place. The SI cDNA in this phenotype revealed 3 mutations, 2 of which, Val to Phe at residue 15 and Ala to Thr at residue 231, had no effect on the structure or function of SI. By contrast, the third mutation resulted in an exchange of leucine by proline at position 620 (L620P) and revealed in transfected COS cells structural features and subcellular localization similar to the phenotype identified in the patient's enterocytes. CONCLUSIONS: This is the first identification at the molecular and subcellular levels of a novel variant of CSID in which SI accumulates predominantly in the ER, and a minor proportion is further processed and transported to the apical membrane of enterocytes.

Vitamin A deficiency interferes with proliferation and maturation of cells in the chicken small intestine.

1. The effect of vitamin A on the small intestine was examined in vitamin-A-deficient meat-type chickens. 2. Maturation and activity of the small intestinal cells were assayed by detection of proliferating cells with proliferating cells nuclear antigen, goblet cells with Alcian blue, mature cells with alkaline phosphatase and extent of RNA expression with dot blot analysis. 3. Vitamin A deficiency caused hyperproliferation of enterocytes, a decrease in the number of goblet cells, decreased alkaline phosphatase activity and decreased expression of 2 brush-border enzymes. 4. Our findings suggest that the absence of vitamin A interferes with the normal growth rate in chickens because it influences functionality of the small intestine by altering proliferation and maturation of cells in the small intestinal mucosa.

Targeted disruption of the mouse villin gene does not impair the morphogenesis of microvilli.

The small intestine is functionally dependent on the presence of the brush border, a tightly packed array of microvilli that forms the amplified apical surface of absorptive cells. In the core of each microvillus, actin filaments are bundled by two proteins, villin and fimbrin. Previous in vitro studies using antisense approaches indicated that villin plays an important role in the morphogenesis of microvilli. To examine the in vivo consequences of villin deficiency, we disrupted the mouse villin gene by targeted recombination in mouse embryonic stem cells. A beta-galactosidase cDNA was also introduced into the villin locus by the targeting event. Homozygous villin-deficient mice are viable, fertile, and display no gross abnormalities. Intact microvilli are present in the small intestine, colon, kidney proximal tubules, and liver bile canaliculi. Although subtle ultrastructural abnormalities can be detected in the actin cores of small intestinal microvilli, localization of sucrase isomaltase, brush border myosin I, and zonula occludens I to the microvillar surface of the small intestine is normal. Thus, in vivo, villin plays a minor or redundant role in the generation of microvilli in multiple absorptive tissues.

Conversion by Peyer's patch lymphocytes of human enterocytes into M cells that transport bacteria.

The epithelium that lines the gut is impermeable to macromolecules and microorganisms, except in Peyer's patches (PPs), where the lymphoid follicle-associated epithelium (FAE) contains M cells that transport antigens and microorganisms. A cultured system that reproduces the main characteristics of FAE and M cells was established by cultivation of PP lymphocytes with the differentiated human intestinal cell line Caco-2. Lymphocytes settled into the epithelial monolayer, inducing reorganization of the brush border and a temperature-dependent transport of particles and Vibrio cholerae. This model system could prove useful for intestinal physiology, vaccine research, and drug delivery studies.

Association between sucrase-isomaltase and p53 expression in colorectal cancer.

BACKGROUND: Sucrase-isomaltase (SI) is a tissue-based phenotypic marker that is an independent prognostic factor in colorectal cancer (CRC). DF3 and galectin 3 are two other tissue-based markers that are upregulated during neoplastic transformation. Because p53 mutations are acquired during neoplastic progression, we reasoned that alterations in SI and p53 may be associated despite an apparent lack of biological interaction. METHODS: Paraffin sections from 183 patients who underwent surgery at New England Deaconess Hospital (NEDH) between 1965 and 1977 were analyzed first by immunohistochemistry (IHC) for the expression of the markers SI, DF3, and galectin 3, which were scored as absent or present. Paraffin sections from a second group of 59 patients who underwent surgery at NEDH between 1985 and 1992 were analyzed by IHC for the expression of p53 as well as SI, DF3, and galectin 3. p53 nuclear staining was scored as absent or present. Previous work has shown that p53 is mutated in all cells with nuclear staining and in 10% of tumors that are unstained. RESULTS: SI expression was not associated with the expression of either DF3 or galectin 3, and neither DF3 nor galectin 3 were prognostic factors in CRC. None of the phenotypic markers were associated with any of the clinicopathologic variables. However, 21 of 24 p53-positive cases (88%) expressed SI, whereas 15 of 35 p53-negative cases (43%) were also SI negative (p = 0.02, Fisher exact test). p53 expression was not associated with expression of DF3 or galectin 3. CONCLUSIONS: SI expression and p53 mutation are associated significantly in CRC. Although the mechanism underlying such an association in presently unknown, the association may define a subset of patients with a worse prognosis.

Forensic application of organ-specific antigens.

A highly sensitive sandwich enzyme immunoassay for organ-specific antigen is described for use in forensic practice. The sandwich enzyme immunoassays for specific antigens to the liver (LSA), the small intestine (sucrase-isomaltase), and the heart (cardiac troponin I) were developed. High levels of antigen could be detected to exist in forensic materials, and it is clearly possible to differentiate between samples from these stabbed organs and those originating from other stabbed organs. In addition, a sandwich enzyme immunoassay for prostate-specific antigen (gamma-seminoprotein, gamma-sm) was developed for sex discrimination of blood and bloodstains. The ratio of gamma-sm to hemoglobin was significantly higher in male adults than in female adults.

Detection of sucrase-isomaltase in stains on a knife by sandwich enzyme immunoassay to determine penetration of the small intestine in a pig model.

Using 2 kitchen knives, 2 stab wounds were made into the abdomen of a pig that had been sacrificed under sanctioned processing methods of a meat packing plant. One knife wound penetrated the jejunum and the other, the liver. After leaving the knives at room temperature for a week, the stains on the blades were extracted and subjected to a sandwich enzyme immunoassay to detect the presence of sucrase-isomaltase (sucrase-alpha-dextrinase or SI), a major digestive enzyme of the small intestine. Results of this assay revealed that sufficient SI could be detected only on the knife that had penetrated the jejunum. This animal experiment thus suggests the potential usefulness of an SI assay to identify a weapon that has penetrated the small intestine. Further investigation will be pursued to determine whether this detection method is also valid in humans.

Conjugated bile salts regulate turnover of rat intestinal brush border membrane hydrolases.

The mechanisms whereby the conjugated bile salts regulate the activities of the brush border membrane hydrolases and its physiological significance were investigated in rat small intestine, and comparisons were made with the action of pancreatic protease. Rat brush border membrane proteins were metabolically labeled with [35S]methionine, and isolated brush border membrane was incubated with taurocholate or pancreatic elastase. The activity of solubilized hydrolases was assayed and the molecular forms of the hydrolases were examined by SDS-PAGE. The activity and protein bands of alkaline phosphatase and sucrase-isomaltase were solubilized by taurocholate, while alkaline phosphatase was not solubilized by elastase. Solubilized sucrase-isomaltase molecules were proteolytically degraded by elastase, whereas the intact molecule of sucrase-isomaltase was solubilized by taurocholate. Next the physiological role of bile salts in brush border membrane hydrolase turnover were investigated using metabolic labeling of brush border membrane hydrolase and immunoprecipitation in biliary diversion rats. After three days of biliary diversion, a significant increase in alkaline phosphatase activity was observed. Although synthesis of alkaline phosphatase in biliary diversion rats was similar to that observed in control rats, biliary diversion rats showed 1.5-fold slower turnover of alkaline phosphatase when compared with control rats. These results suggest that conjugated bile salts in the intestinal lumen may cause a rapid turnover of brush border membrane hydrolases, which may be increased by the enhanced enzyme degradation. The mechanisms for the enhanced degradation appeared to be solubilization of hydrolases caused by the detergent activity of bile salts. Therefore, conjugated bile salts may play an important physiological role in the regulation of expression of the protease-resistant enzymes such as alkaline phosphatase.

Restriction of lactase gene expression along the proximal-to-distal axis of rat small intestine occurs during postnatal development.

BACKGROUND/AIMS: Developmental changes of lactase activity along the proximal-to-distal axis of the small intestine are poorly understood. A study of delineate lactase gene expression at the cellular level was undertaken. METHODS: The topographical regulation of lactase was studied in conjunction with sucrase-isomaltase in proximal, middle, and distal segments of 0-, 7-, 14-, 16-, 18-, 21-, and 28-day-old and adult rats using in sity hybridization, immunohistochemistry, and ribonuclease protection assays. RESULTS: From 0 to 16 days, lactase messenger RNA (mRNA) and protein were abundant along the total length of the small intestine. However, at weaning, lactase mRNA and protein were no longer detectable in the terminal ileum. After 28 days, zones of reduced lactase expression were found in the duodenum and terminal ileum. These zones demonstrated expression of lactase protein in scattered enterocytes along the villus (patchy expression). In contrast, sucrase-isomaltase was first detected at 16 days, with patchy expression along the total small intestine; at 21 days it was abundant. CONCLUSIONS: Concordant changes in both lactase mRNA and protein detection during development suggest that the horizontal gradient of lactase enzyme expression is dependent on lactase mRNA abundance. Furthermore, zones of patchy lactase expression appear around weaning and flank the area of high lactase expression in the midintestine. Patchy expression is also found for sucrase-isomaltase before weaning.

Sucrase-isomaltase gene expression in Barrett's esophagus and adenocarcinoma.

BACKGROUND: Specialized Barrett's esophageal mucosa, characterized by incomplete intestinal metaplasia of the esophageal mucosa, is associated with the development of adenocarcinoma. Although the intestinal disaccharidase sucrase-isomaltase (SI) has been shown in incomplete intestinal metaplasia of the stomach, it is commonly believed that Barrett's mucosa does not express SI based on the lack of enzymatic activity. This study was undertaken to determine whether the SI gene is expressed in Barrett's epithelium and its associated adenocarcinoma at the level of messenger RNA (mRNA) and protein. METHODS: Reverse transcription polymerase chain reaction was used to determine the presence of SI mRNA in Barrett's esophagus and esophageal adenocarcinomas. Cellular localization of SI protein was determined by immunohistochemistry. RESULTS: SI mRNA was identified in 76% of Barrett's epithelium and 82% of esophageal adenocarcinomas. The transcriptional initiation site for SI in these tissues was identical to that of the small intestine. Immunohistochemical localization showed that SI was directed to the apical membrane in Barrett's epithelium in contrast to a more diffuse cytoplasmic pattern in esophageal adenocarcinomas. CONCLUSIONS: Columnar cells of specialized Barrett's epithelium express SI and are, therefore, phenotypically similar to those in incomplete intestinal metaplasia of the stomach with respect to intestinal gene expression.

Immunoelectron microscopic localization of sucrase-isomaltase in rat small intestine.

The localization of rat small intestinal microvillous sucrase-isomaltase was studied by immunoelectron microscopy to investigate its intracellular transport to the microvillous membrane. At the same time, the usefulness of a rapid embedding method of tissues in Lowicryl K4M for immunocytochemistry of sucrase-isomaltase was examined. Sucrase-isomaltase was present not only in the microvillous membrane, but also in the apical vesicles and the apical plasma membrane invaginations. Negligible labeling was observed in the other portions of the absorptive cells. These findings suggest that the final step of intracellular transport of sucrase-isomaltase to the microvillous membrane is via smooth apical vesicles. The rapid immunoelectron microscopic method adopted in this study seemed to be a useful technique for the study of the intracellular localization of sucrase-isomaltase.

Transient mosaic patterns of morphological and functional differentiation in the Caco-2 cell line.

To gain further insight on the mosaic expression of specific functional intestinal markers (such as sucrase-isomaltase) in postconfluent Caco-2 cells, a human colon cancer cell line unique in its property to differentiate in vitro into a mature enterocyte-like cell type, a comparative study was undertaken to examine the morphological and functional differentiation of Caco-2 cells at various culture stages. The observations clearly indicate that Caco-2 cells can exist only in three different states in culture: homogeneously undifferentiated (at subconfluence), heterogeneously polarized and differentiated (between 0 and 20 days after confluence), and homogeneously polarized and differentiated (after 30 days). Indeed, in the intermediate state, a strong discrepancy is found among adjacent differentiating cells throughout the monolayer relative to sucrase-isomaltase expression as well as to cell morphology and brush border organization. Back-scattered electron imaging analysis showed a lack of correlation between these parameters at the cellular level. These observations indicate that morphological and functional differentiations of Caco-2 cells progress concomitantly according to a transient mosaic pattern, thus providing evidence that these two processes are not coupled.

Sucrase-isomaltase expression in chronic ulcerative colitis and dysplasia.

Sucrase-isomaltase (SI) is a mucosal disaccharidase that is present in normal small intestine and fetal colon. It also has been noted in colonic adenomas and adenocarcinomas. We used a polyclonal antibody to human SI to investigate enzyme presence and utility in detecting dysplastic changes in chronic ulcerative colitis. Sections from 32 cases were reviewed for the presence or absence of active colitis and dysplasia. Immunostaining of these cases for SI was performed and the results were reported based on location of immunoreactivity (ie, membrane and cytoplasmic staining in superficial and crypt epithelial cells) and percentage of positivity. Of 81 sections examined, 48 were rated negative for dysplasia (23 inactive colitis, 20 active, and five probably negative) and 28 were rated positive (eight low grade and 20 high grade). Surface membrane staining of epithelial cells was noted in all 28 dysplastic slides and positive cases (sensitivity, 100%) but also in 29 of 48 negative sections (P less than .001). In contrast, cytoplasmic positivity was present in 25 of 28 dysplastic and in only two of 48 negative slides (P less than .0001). The presence of cytoplasmic staining of SI in the superficial or crypt cells revealed a sensitivity of 92% and a specificity of 94%. There were five additional sections rated as indefinite for dysplasia (probably positive or unknown); two showed staining patterns typical of negative slides and three showed positive staining patterns. Of the 18 samples of transitional mucosa next to areas of dysplasia, surface membrane staining of SI was seen in all samples and cytoplasmic staining was seen in 15. We conclude that membrane staining of SI can be detected in inflammatory, regenerative, and dysplastic mucosa in ulcerative colitis. Cytoplasmic staining, however, correlates strongly with the presence of dysplastic change and may help in its detection.

Expression of sucrase-isomaltase mRNA along the villus-crypt axis in the rat small intestine.

Sucrase-isomaltase has been used as a marker enzyme to study cell differentiation along the intestinal villus-crypt axis. Previous studies are in agreement that sucrase activity is confined to villus epithelial cells. However, immunoreactivity data are at conflict, with some studies reporting sucrase antigen in crypts as well as villi. To resolve this discrepancy, our goal was to determine the distribution of sucrase-isomaltase mRNA. A cDNA clone representing 3.0 kb of rat sucrase-isomaltase, including the sucrase active site, was characterized. Northern analysis of 12 tissues demonstrated a 6 kb transcript only in the small intestine. Jejunal cell fractions prepared by a washing technique showed declining levels of both sucrase activity and sucrase-isomaltase mRNA as well as increasing levels of thymidine kinase activity from early to later fractions. Since later fractions did not yield pure crypt cells, in situ hybridization using an 35S-labeled sucrase-isomaltase riboprobe was performed. The transition from zero to intense signal at the crypt-villus junction leads us to conclude that in the adult rat, sucrase-isomaltase gene expression is initiated only after cells leave the proliferative cycle and migrate onto the villi.

Intestinal brush-border-associated enzymes: co-ordinated expression in colorectal cancer.

The brush border of normal small-intestine epithelial cells is rich in enzymes that are involved in the digestive process. Such molecules can be used as markers to analyze cell lineages and differentiation properties of colorectal cancers. Monoclonal antibodies detecting dipeptidyl peptidase-IV, aminopeptidase N, endopeptidase F, sucrase-isomaltase, alkaline phosphatase, maltase-glucoamylase and lactase have been used to analyze the phenotype of colorectal cancers, adjacent mucosa and histologically normal distant mucosa. The avidin-biotin peroxidase complex method was used. Expression of dipeptidyl peptidase-IV, aminopeptidase N, sucrase-isomaltase and alkaline phosphatase was common in non-neoplastic mucosa adjacent to, and distant from, the tumor; in contrast, endopeptidase F, maltase-glucoamylase and lactase were rarely expressed in normal distant mucosa and more frequently expressed in mucosa adjacent to the tumor. Dipeptidyl peptidase-IV, aminopeptidase N, endopeptidase F, sucrase-isomaltase and alkaline phosphatase were frequently expressed in colorectal cancers, whereas maltase-glucoamylase and lactase were rarely expressed. Two general patterns of antibody reactivity were observed: diffuse cytoplasmic and apical; apical reactivity was generally associated with more differentiated tumors. A logistic predictive regression model indicated that enzyme expression in colorectal cancers followed a coordinate pattern, but was unrelated to the location of the tumor, Dukes stage or differentiation grade. In conclusion, expression of brush-border-associated enzymes occurs frequently in colorectal cancers and is regulated in a co-ordinated manner. These markers can be used for the phenotypic sub-classification of colorectal cancers.

Expression and different polarity of aminopeptidase N in normal human colonic mucosa and colonic tumors.

Expression and cellular localization of brush-border enzymes (aminopeptidase N, dipeptidylpeptidase IV, lactase, maltase) in normal human colon, colonic polyps and malignant intestinal tumors were investigated with a panel of monoclonal antibodies reacting with either native or denatured proteins. The enzymes were detected on cryostat sections by indirect immunofluorescence staining, or affinity-purified and analyzed by gel electrophoresis and immunoblotting. Dipeptidylpeptidase IV, lactase and maltase were absent from all samples examined, while aminopeptidase N (APN) was detected at the basal membrane of the epithelial cells in most specimens of colon obtained from individuals free of intestinal tumors. In contrast, APN was frequently localized at the luminal membrane of the surface epithelium in large-intestinal mucosa distal to tumors, adenomas and hyperplastic polyps, and from members of hereditary colon cancer syndrome families. APN was also expressed in colonic tumors, where it was present in an apical cell membrane location in 3/23 adenomas and 14/35 adenocarcinomas examined. No correlation was found between tumor-cell invasiveness (classified by "Dukes" stage) and expression or cellular location of aminopeptidase N. Histologically, all positive tumors were moderately or well differentiated. These results suggest that aminopeptidase N is normally expressed in adult human colon, but epithelial cells in the large and small intestine differ in their ways of sorting this enzyme intracellularly and eventually inserting it into different aspects of their surface membrane, a process which may be altered at an early stage of carcinogenesis.

[Do immunologic markers facilitate differentiation between histologic types of stomach cancer?]. / Ermöglichen immunologische Marker eine Differenzierung zwischen den histologischen Typen des Magenkarzinoms?

Seventeen gastric carcinomas (intestinal n = 12; diffuse n = 1; mixed type n = 4) and one Barrett's carcinoma were prospectively studied by immunohistochemistry for the expression of different keratin polypeptides and of the brush border markers villin, sucrase isomaltase and aminopeptidase N. All carcinomas expressed the keratin polypeptides 8, 18, and 19 and were stained by the broad specific keratin antibody KL1, irrespective of histologic type. Keratin 7, however, was expressed in only one carcinoma in most tumor cells and in two further carcinomas in some tumor cells. Thus, specific differentiation of the various histologic types of gastric carcinoma does not seem to be aided by the use of keratin antibodies. Villin was positive in 80% of the tumors and sucrase isomaltase and aminopeptidase N were positive in 67% respectively with no obvious histologic difference. The frequent positivity of the brush border markers, usually typical for intestinal epithelium, reflects the high degree of intestinal differentiation of gastric carcinomas, but again does not seem to be associated with a particular histologic type.